
Simultaneous Application of Methylene Blue and Chlorin e6 Photosensitizers: Investigation on a Cell Culture
The application of photosensitizers for inhibition of oxidative phosphorylation in order to temporally decrease oxygen uptake by tumor cells in the course of photodynamic therapy (PDT) evokes growing interest.
The aim of the study is to overcome tumor hypoxia for further photodynamic therapy with simultaneous use of type I photosensitizer methylene blue (MB) and type II photosensitizer chlorin e6.
Material and Methods. A photodynamic activity of MB and its combined use with chlorin e6 has been studied on the HeLa cell culture, their effect on cell metabolism in their co-accumulation and subsequent irradiation has also been assessed.
Results. MB generates reactive oxygen species in the cells in contrast to chlorin e6, which produces singlet oxygen. Besides, MB is converted to a colorless leucoform at low concentrations in the process of de-oxygenation. Incubation of cells with MB concurrently with chlorin e6 results in its greater fluorescence as compared to the incubation with MB only. MB concentration in the range of 1–10 mg/kg and the laser radiation dose of 60 J/cm2 do not cause cell death, probably, due to the MB transition to the photodynamically inactive leucoform. Cell death is observed after PDT in all samples with chlorin e6 and with MB at the 0–20 mg/kg concentration ranges and at 60 J/cm2 radiation dose. The phototoxicity of MB together with chlorin e6 is higher than that of chlorin e6 alone. The analysis of metabolic NADH cofactor lifetime after the incubation of the cells with MB and chlorin e6, and after PDT with them has revealed the presence of stress seen as an extension of NADH fluorescence cloud along the metabolic axis. After PDT with low concentrations of MB, the NADH fluorescent cloud on the phasor diagram shifts to the right towards short lifetimes (closer to anaerobic glycolysis along the NADH metabolic trajectory). The PDT with MB and chlorin e6 leads to the shift of the NADH fluorescence cloud on the phasor diagram to the left towards long lifetimes (closer to oxidative phosphorylation along the NADH metabolic trajectory). In this case, the cells die due to necrosis.
Conclusion. The co-accumulation of MB with chlorin e6 prevents MB reduction to a colorless leucoform, decreasing the oxygen uptake by the cells and making it possible to use simultaneously type I and II photodynamic reactions.
- Du J., Shi T., Long S., Chen P., Sun W., Fan J., Peng X. Enhanced photodynamic therapy for overcoming tumor hypoxia: from microenvironment regulation to photosensitizer innovation. Coordination Chemistry Reviews 2021; 427, 213604, https://doi.org/10.1016/j.ccr.2020.213604.
- Wan Y., Fu L.H., Li C., Lin J., Huang P. Conquering the hypoxia limitation for photodynamic therapy. Adv Mater 2021; 33(48): e2103978, https://doi.org/10.1002/adma.202103978.
- Li X., Chen L., Huang M., Zeng S., Zheng J., Peng S., Wang Y., Cheng H., Li S. Innovative strategies for photodynamic therapy against hypoxic tumor. Asian J Pharm Sci 2023; 18(1): 100775, https://doi.org/10.1016/j.ajps.2023.100775.
- Vander Heiden M.G., Cantley L.C., Thompson C.B. Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 2009; 324(5930): 1029–1033, https://doi.org/10.1126/science.1160809.
- Su Y., Lu K., Huang Y., Zhang J., Sun X., Peng J., Zhou Y., Zhao L. Targeting Warburg effect to rescue the suffocated photodynamic therapy: a cancer-specific solution. Biomaterials 2023; 294: 122017, https://doi.org/10.1016/j.biomaterials.2023.122017.
- Gao F., Dong J.H., Xue C., Lu X.X., Cai Y., Tang Z.Y., Ou C.J. Tumor-targeting multiple metabolic regulations for bursting antitumor efficacy of chemodynamic therapy. Small 2024; 20(26): e2310248, https://doi.org/10.1002/smll.202310248.
- Yuan P., Deng F.A., Liu Y.B., Zheng R.R., Rao X.N., Qiu X.Z., Zhang D.W., Yu X.Y., Cheng H., Li S.Y. Mitochondria targeted O2 economizer to alleviate tumor hypoxia for enhanced photodynamic therapy. Adv Healthc Mater 2021; 10(12): e2100198, https://doi.org/10.1002/adhm.202100198.
- Chen Q., Chen J., Liang C., Feng L., Dong Z., Song X., Song G., Liu Z. Drug-induced co-assembly of albumin/catalase as smart nano-theranostics for deep intra-tumoral penetration, hypoxia relieve, and synergistic combination therapy. J Control Release 2017; 263: 79–89, https://doi.org/10.1016/j.jconrel.2016.11.006.
- Colegio O.R., Chu N.Q., Szabo A.L., Chu T., Rhebergen A.M., Jairam V., Cyrus N., Brokowski C.E., Eisenbarth S.C., Phillips G.M., Cline G.W., Phillips A.J., Medzhitov R. Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature 2014; 513(7519): 559–563, https://doi.org/10.1038/nature13490.
- Huber V., Camisaschi C., Berzi A., Ferro S., Lugini L., Triulzi T., Tuccitto A., Tagliabue E., Castelli C., Rivoltini L. Cancer acidity: an ultimate frontier of tumor immune escape and a novel target of immunomodulation. Semin Cancer Biol 2017; 43: 74–89, https://doi.org/10.1016/j.semcancer.2017.03.001.
- Jiang H., Jedoui M., Ye J. The Warburg effect drives dedifferentiation through epigenetic reprogramming. Cancer Biol Med 2024; 20(12): 891–897, https://doi.org/10.20892/j.issn.2095-3941.2023.0467.
- Komlódi T., Tretter L. Methylene blue stimulates substrate-level phosphorylation catalysed by succinyl-CoA ligase in the citric acid cycle. Neuropharmacology 2017; 123: 287–298, https://doi.org/10.1016/j.neuropharm.2017.05.009.
- Xue H., Thaivalappil A., Cao K. The potentials of methylene blue as an anti-aging drug. Cells 2021; 10(12): 3379, https://doi.org/10.3390/cells10123379.
- Pominova D., Ryabova A., Skobeltsin A., Markova I., Linkov K., Romanishkin I. The use of methylene blue to control the tumor oxygenation level. Photodiagnosis Photodyn Ther 2024; 46: 104047, https://doi.org/10.1016/j.pdpdt.2024.104047.
- Bouillaud F., Ransy C., Moreau M., Benhaim J., Lombès A., Haouzi P. Methylene blue induced O2 consumption is not dependent on mitochondrial oxidative phosphorylation: implications for salvage pathways during acute mitochondrial poisoning. Respir Physiol Neurobiol 2022; 304: 103939, https://doi.org/10.1016/j.resp.2022.103939.
- Balcerczyk A., Damblon C., Elena-Herrmann B., Panthu B., Rautureau G.J.P. Metabolomic approaches to study chemical exposure-related metabolism alterations in mammalian cell cultures. Int J Mol Sci 2020; 21(18): 6843, https://doi.org/10.3390/ijms21186843.
- Ranjit S., Malacrida L., Jameson D.M., Gratton E. Fit-free analysis of fluorescence lifetime imaging data using the phasor approach. Nat Protoc 2018; 13(9): 1979–2004, https://doi.org/10.1038/s41596-018-0026-5.
- Alhayaza R., Haque E., Karbasiafshar C., Sellke F.W., Abid M.R. The relationship between reactive oxygen species and endothelial cell metabolism. Front Chem 2020; 8: 592688, https://doi.org/10.3389/fchem.2020.592688.
- Junqueira H.C., Severino D., Dias L.G., Gugliotti M.S., Baptista M.S. Modulation of methylene blue photochemical properties based on adsorption at aqueous micelle interfaces. PCCP 2002; 11: 2320–2328, https://doi.org/10.1039/b109753a.
- Fernández-Pérez A., Marbán G. Visible light spectroscopic analysis of methylene blue in water; what comes after dimer? ACS Omega 2020; 5(46): 29801–29815, https://doi.org/10.1021/acsomega.0c03830.
- Dean J.C., Oblinsky D.G., Rather S.R., Scholes G.D. Methylene blue exciton states steer nonradiative relaxation: ultrafast spectroscopy of methylene blue dimer. J Phys Chem B 2016; 120(3): 440–454, https://doi.org/10.1021/acs.jpcb.5b11847.
- Kalinina S., Freymueller C., Naskar N., von Einem B., Reess K., Sroka R., Rueck A. Bioenergetic alterations of metabolic redox coenzymes as NADH, FAD and FMN by means of fluorescence lifetime imaging techniques. Int J Mol Sci 2021; 22(11): 5952, https://doi.org/10.3390/ijms22115952.
- Ranjit S., Malacrida L., Stakic M., Gratton E. Determination of the metabolic index using the fluorescence lifetime of free and bound nicotinamide adenine dinucleotide using the phasor approach. J Biophotonics 2019; 12(11): e201900156, https://doi.org/10.1002/jbio.201900156.
- Vermathen M., Vermathen P., Simonis U., Bigler P. Time-dependent interactions of the two porphyrinic compounds chlorin e6 and mono-L-aspartyl-chlorin e6 with phospholipid vesicles probed by NMR spectroscopy. Langmuir 2008; 24(21): 12521–12533, https://doi.org/10.1021/la802040v.
- Vermathen M., Marzorati M., Vermathen P., Bigler P. pH-dependent distribution of chlorin e6 derivatives across phospholipid bilayers probed by NMR spectroscopy. Langmuir 2010; 26(13): 11085–11094, https://doi.org/10.1021/la100679y.
- Marzorati M., Bigler P., Vermathen M. Interactions between selected photosensitizers and model membranes: an NMR classification. Biochim Biophys Acta 2011; 1808(6): 1661–1672, https://doi.org/10.1016/j.bbamem.2011.02.011.
- Vermathen M., Kämpfer T., Nuoffer J.M., Vermathen P. intracellular fate of the photosensitizer chlorin e4 with different carriers and induced metabolic changes studied by 1H NMR spectroscopy. Pharmaceutics 2023; 15(9): 2324, https://doi.org/10.3390/pharmaceutics15092324.
- Gabrielli D., Belisle E., Severino D., Kowaltowski A.J., Baptista M.S. Binding, aggregation and photochemical properties of methylene blue in mitochondrial suspensions. Photochem Photobiol 2004; 79(3): 227–232, https://doi.org/10.1562/be-03-27.1.
- Iqbal M.J., Kabeer A., Abbas Z., Siddiqui H.A., Calina D., Sharifi-Rad J., Cho W.C. Interplay of oxidative stress, cellular communication and signaling pathways in cancer. Cell Commun Signal 2024; 22(1): 7, https://doi.org/10.1186/s12964-023-01398-5.
- Rusz M., Del Favero G., El Abiead Y., Gerner C., Keppler B.K., Jakupec M.A., Koellensperger G. Morpho-metabotyping the oxidative stress response. Sci Rep 2021; 11(1): 15471, https://doi.org/10.1038/s41598-021-94585-8.
- Argüello R.J., Combes A.J., Char R., Gigan J.P., Baaziz A.I., Bousiquot E., Camosseto V., Samad B., Tsui J., Yan P., Boissonneau S., Figarella-Branger D., Gatti E., Tabouret E., Krummel M.F., Pierre P. SCENITH: a flow cytometry-based method to functionally profile energy metabolism with single-cell resolution. Cell Metab 2020; 32(6): 1063–1075.e7, https://doi.org/10.1016/j.cmet.2020.11.007.
- Miskolci V., Tweed K.E., Lasarev M.R., Britt E.C., Walsh A.J., Zimmerman L.J., McDougal C.E., Cronan M.R., Fan J., Sauer J.D., Skala M.C., Huttenlocher A. In vivo fluorescence lifetime imaging of macrophage intracellular metabolism during wound responses in zebrafish. Elife 2022; 11: e66080, https://doi.org/10.7554/eLife.66080.
- Datta R., Alfonso-García A., Cinco R., Gratton E. Fluorescence lifetime imaging of endogenous biomarker of oxidative stress. Sci Rep 2015; 5: 9848, https://doi.org/10.1038/srep09848.
- Shi D.Y., Xie F.Z., Zhai C., Stern J.S., Liu Y., Liu S.L. The role of cellular oxidative stress in regulating glycolysis energy metabolism in hepatoma cells. Mol Cancer 2009; 8: 32, https://doi.org/10.1186/1476-4598-8-32.
- Leben R., Köhler M., Radbruch H., Hauser A.E., Niesner R.A. Systematic enzyme mapping of cellular metabolism by phasor-analyzed label-free NAD(P)H fluorescence lifetime imaging. Int J Mol Sci 2019; 20(22): 5565, https://doi.org/10.3390/ijms20225565.